Antithrombotic dual inhibitors comprising a biotin residue

ABSTRACT

The present invention relates compounds of the formula (I) oligosaccharide-spacer-A (I), wherein the oligosaccharide is a negatively charged oligosaccharide residue comprising two to twenty five monosaccharide units, the charge being compensated by positively charged counterions, and wherein the oligosaccharide residue is derived from an oligosaccharide which has (AT-III mediated) anti-Xa activity per se; the spacer is an essentially pharmacologically inactive flexible linking residue having a chain length of 10 to 70 atoms; A is the residue —CH[NH—SO 2 —R 1 ] [CO—NR 2 —CH(4-benzamidine)-CO—NR 3 R 4 ], wherein R1 is phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, (iso)quinolinyl, tetrahydro(iso)quinolinyl, 3,4-dihydro-1H-isoquinolinyl, chromanyl or the camphor group, which groups may optionally be substituted with one or more substituents selected from (1-8C)alkyl or (1-8C)alkoxy; and wherein R 2  and R 3  are independently H or (1-8C)alkyl; R 4  is (-8C)alkyl or (3-8C)cycloalkyl; or R 3  and R 4  together with the nitrogen atom to which they are bonded are a nonaromatic (4-8)membered ring optionally containing another heteroatom, the ring optionally being substituted with (1-8C)alkyl or SO2-(I-8C)alkyl; or a pharmaceutically acceptable salt thereof a prodrug or solvate thereof; wherein the compound of formula I further comprises at least one covalent bond with a biotin residue or an analogue thereof. The compounds of the invention have antithrombotic activity and can be used in treating or preventing thrombosis or other thrombin-related diseases. The antithrombotic activity of the compound of this invention can be neutralized in case of emergency upon administration of avidin, streptavidin and analogues thereof having high biotin affinity.

The present invention relates to new antithrombotic dual inhibitorscomprising a biotin residue or a biotin derivative, a process for theirpreparation, pharmaceutical compositions containing the compounds asactive ingredients, as well as the use of said compounds for themanufacture of medicaments.

Recent progress in the search for synthetic active pharmaceuticalsubstances having similar or superior antithrombotic properties whencompared to heparin, has resulted in the design of new dual inhibitorse.g. as described in WO 99/65934 and WO 01/42262. Those compounds aretypically conjugates of an oligosaccharide residue connected to a directthrombin inhibitor by an essentially pharmacologically inactive spacer.The oligosaccharide residue in the molecule displays anti-thrombin III(AT-III) mediated anti-Xa activity. Thus, the new conjugates have dual,antithrombotic and anticoagulant, activity.

An excellent example of the new class of dual inhibitors is the compoundindicated with the code name Org 42675, in which a pentasaccharide islinked to a direct inhibitor of thrombin, having the followingstructure:

Studies in experimental thrombosis have demonstrated that this compound,in addition to potent anticoagulant and antithrombotic properties, alsoinhibits the activity of clot-bound thrombin. Further, Org 42675appeared to be highly efficacious in the prevention of thromboticreocclusion following thrombolysis of occlusive arterial thrombi. Thecompound displays a 10-fold prolonged half-life in comparison to thecorresponding non-conjugated direct thrombin inhibitor derived fromNAPAP. In comparison with argatroban, heparin and fondaparinux, Org42675 showed improved efficacy. (Journal of Thrombosis and Haemostasis,Volume 1, Issue 9, Page 1945, 2003).

The clinical potential of the new dual inhibitors is considered to besignificant and therefore clinical testing has already been initiated.

As a precautionary measure, within the field of anticoagulant andantithrombotic therapy, there is a need for an antidote to be able toeffectively neutralize or minimize the activity of the anticoagulant orantithrombotic drug used. This is because it is well known that ahaemorrhage can be triggered in a patient under treatment for anyaccidental cause. Further, it may be necessary to intervene surgicallyin a patient under antithrombotic or anticoagulant treatment. Inaddition, during some surgical procedures, anticoagulants may be used ata high dose so as to prevent blood coagulation and it is necessary toneutralize them at the end of the operation. It is thereforeadvantageous to have antithrombotic/anticoagulant agents available whichcan be neutralized in order to stop the antithrombotic/anticoagulantactivity at any time.

In US 2004/0024197 it is disclosed that, in case of emergency, theantithrombotic activity of certain polysaccharides may be reduced usingavidin, if those polysaccharides contain at least a covalent bond withbiotin or a biotin derivative.

The present invention relates to novel neutralizable dual inhibitorsderived from the dual inhibitors described in WO 99/65934 and WO01/42262. It has been found that a certain biotin “label”, being thegroup

also referred to in this document as “BT” (derived fromhexahydro-2-oxo-1H-thieno[3,4-d]imidazole-4-pentanoic acid, preferablythe D(+)-isomer) or an analogue thereof, can be attached to orintroduced into the structure of the compounds described in WO 99/65934and WO 01/42262, resulting in neutralizable dual inhibitors.

Thus, the present invention relates to compounds of the formula (I)

oligosaccharide-spacer-A  (I),

wherein the oligosaccharide is a negatively charged oligosaccharideresidue comprising two to twenty five monosaccharide units, the chargebeing compensated by positively charged counterions, and wherein theoligosaccharide residue is derived from an oligosaccharide which has(AT-III mediated) anti-Xa activity per se;the spacer is an essentially pharmacologically inactive flexible linkingresidue having a chain length of 10 to 70 atoms;A is the residue —CH[NH—SO₂—R¹][CO—NR²—CH(4-benzamidine)-CO—NR³R⁴],wherein R¹ is phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl,(iso)quinolinyl, tetrahydro(iso)quinolinyl,3,4-dihydro-1H-isoquinolinyl, chromanyl or the camphor group, whichgroups may optionally be substituted with one or more substituentsselected from (1-8C)alkyl or (1-8C)alkoxy; and wherein R² and R³ areindependently H or (1-8C)alkyl; R⁴ is (1-8C)alkyl or (3-8C)cycloalkyl;or R³ and R⁴ together with the nitrogen atom to which they are bondedare a nonaromatic (4-8)membered ring optionally containing anotherheteroatom, the ring optionally being substituted with (1-8C)alkyl orSO₂-(1-8C)alkyl;or a pharmaceutically acceptable salt thereof or a prodrug or solvatethereof;wherein the compound of formula I further comprises at least onecovalent bond with a biotin residue or an analogue thereof.

The compounds of the invention are dual inhibitors, having a tuneablemixed profile of both non-mediated, direct anti-thrombin (factor IIa)activity And anti-thrombin III (AT-III) mediated anti-Xa activity. Themixed profile of the compounds of the invention may be tuned by varyingthe nature of the oligosaccharide residue and the potency of the directthrombin inhibitor (NAPAP analogues). A range of profiles is therebyavailable. Compounds of the invention have a long plasma half-life and,as a result, they possess prolonged anti-thrombin activity compared toNAPAP or its derivatives reported in literature previously. In addition,compounds of the invention may escape the neutralizing action ofplatelet factor 4 (PF4). Low toxicity is also an advantageous aspect ofcompounds of this invention.

The compounds of the present invention are useful for treating andpreventing thrombin-mediated and thrombin-associated diseases. Thisincludes a number of thrombotic and prothrombotic states in which thecoagulation cascade is activated which include, but are not limited to,deep vein thrombosis, pulmonary embolism, thrombophlebitis, arterialocclusion from thrombosis or embolism, arterial reocclusion during orafter angioplasty or thrombolysis, restenosis following arterial injuryor invasive cardiological procedures, postoperative venous thrombosis orembolism, acute or chronic atherosclerosis, stroke, myocardialinfarction, cancer and metastasis, and neurodegenerative diseases. Thecompounds of the invention may also be used as anticoagulants inextracorporeal blood circuits, as necessary in dialysis and surgery. Thecompounds of the invention may also be used as in vitro anticoagulants.

The biotin label (or analogue thereof) in the compound of the presentinvention is rapidly recognized by and binds to a specific antidote,being avidin (The Merck Index, Twelfth edition, 1996, M.N. 920, pages151-152) or streptavidin, two tetrameric proteins with respective massesequal to approximately 66 000 and 60 000 Da which have a very highaffinity for biotin. Thus, in an emergency situation, the action of thedual inhibitor can be rapidly neutralized by using avidin orstreptavadin, for example by injection of a pharmaceutical solutioncontaining the same. Analogues of avidin and streptavidin having highbiotin affinity may be used similarly. The resulting inactiveantidote-inhibitor complex is cleared from the blood circulation.

Biotin analogues, which may be used as a label according to thisinvention, may be selected from, but are not limited to, the biotinanalogues shown in the Pierce catalogue, 1999-2000, pages 62 to 81, forexample 6-biotinamidohexanoate, 6-(6-biotinamidohexanamido)hexanoate,and 2-biotinamidoethanethiol, etc. In such analogues, the biotin residueBT, as previously defined, is a characteristic part of the molecule.Other analogues are for example biotin analogues that are alkyated atthe biotinamide bond (wherein alkyl is (1-4C)alkyl, preferably methyl)and which are stable to biotinidase cleavage (Bioconjugate Chem., Vol.11, 2000, 569-583; Bioconjugate Chem., Vol. 11, 2000, 584-598) or otherbiotin analogues comprising for example a hydroxymethylene, carboxylate,or acetate alpha to the biotinamide bond, such as described inBioconjugate Chem., Vol. 12, No. 4, 2001, 616-623.

Preferred residues of biotin analogues have the formula—(NH—CO)_(n)—(CH₂)_(p)—NR-BT, wherein n is 0 or 1 (in particular n is0), p is 4 or 5 (in particular p is 4), R═H or (1-4C)alkyl and BT is aspreviously defined. In preferred embodiments, R is H.

It has been found in comparative studies with their correspondingnon-biotinylated compounds that the introduction of a biotin label intothe dual inhibitors of this invention does essentially not interferewith their direct thrombin inhibitory potency nor with theiranti-thrombin III (AT-III) mediated anti-Xa activity. In addition, theantithrombotic activity of the compounds of formula I is (essentially)completely neutralized upon administration of avidin or streptavidin.

Any negatively charged oligosaccharide residue of two to twenty fivemonosaccharide units is usable in the compounds of the presentinvention. Suitable compounds of the invention are compounds wherein theoligosaccharide is a sulfated oligosaccharide residue. Preferably, theoligosaccharide residue is derived from an oligosaccharide which has(AT-III mediated) anti-Xa activity per se, such as the oligosaccharidesdisclosed in EP 0,454,220, EP 0,529,715, WO 97/47659, WO 98/03554, WO99/36443 and WO 99/36428. Further preferred are oligosaccharide residueshaving two to sixteen, in particular two to six, monosaccharide units.Most preferably the oligosaccharide is a sulfated pentasaccharideresidue. Preferred pentasaccharide residues have the formula (II)

wherein R⁵ is independently a biotin residue or analogue thereof, OSO₃ ⁻or (1-8C)alkoxy. In preferred pentasaccharide residues, the total numberof sulfate groups is 4, 5, 6 or 7.

Preferred compounds according to the invention are compounds wherein thepentasaccharide residue has the structure:

wherein R⁵ is OCH₃ or OSO₃ ⁻.

An in particular preferred pentasaccharide residue is

Further preferred compounds of the invention are compounds of formula I,wherein R¹ is phenyl or naphthyl, optionally substituted with one ormore substituents selected from methyl or methoxy. More preferred, R¹ is4-methoxy-2,3,6-trimethylphenyl. In preferred compounds, NR³R⁴represents the piperidinyl group. Preferably, R² is H.

The spacer is an essentially pharmacologically inactive flexible linkingresidue, preferably having 10-50 atoms counted along the “backbone” ofthe spacer, the oxygen of the oligosaccharide residue not included.Further preferred is a length of 13-25 atoms, preferably 16-22, and mostpreferred 19 atoms.

The chemical nature of the spacer is of minor importance for theanti-thrombotic activity of the compounds of the invention. The spacermay comprise (somewhat) rigid elements, such as ring structures andunsaturated bonds. Highly flexible spacers are more suitable thanothers. Suitable spacers may easily be designed by a person skilled inthe art. For synthetic reasons longer spacers are considered lesssuitable, however, longer spacers may still successfully be applied inthe compounds of the present invention. Preferred spacers comprise atleast one —(CH₂CH₂O)-element.

Preferred compounds according to the invention comprise one covalentbond with a biotin residue or analogue thereof.

The biotin (or analogue thereof) label may be present in all parts ofthe compound formula I. Therefore, embodiments of this invention arecompounds wherein (a) the oligosaccharide residue of the compound offormula I comprises a covalent bond with a biotin residue or analoguethereof, (b) the spacer of the compound of formula I comprises acovalent bond with a biotin residue or analogue thereof and (c) theresidue A of the compound of formula I comprises a covalent bond with abiotin residue or analogue thereof (which means that a hydrogen atom orsubstituent in the definition of A has been replaced by the biotinresidue).

Representative examples of the biotinylated dual inhibitors of thepresent invention are

and salts thereof, but also compounds of formula I, wherein the spaceris attached to the oligosaccharide at another position, and/or compoundswherein the biotin (analogue) residue is present at other positions ofthe molecule. Preferred is the sodium salt.

The compound of formula III is a preferred example of this invention.

In the description of the compounds of formula (I) the followingdefinitions are used.

The terms (1-4C)alkyl and (1-8C)alkyl mean a branched or unbranchedalkyl group having 1-4 and 1-8 carbon atoms, respectively, for examplemethyl, ethyl, propyl, isopropyl, butyl, sec-butyl, tert-butyl, hexyland octyl. Methyl and ethyl are preferred alkyl groups.

The term (1-8C)alkoxy means an alkoxy group having 1-8 carbon atoms, thealkyl moiety having the meaning as previously defined. Methoxy is apreferred alkoxy group.

The term (3-8C)cycloalkyl means a cycloalkyl group having 3-8 carbonatoms, being cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl or cyclo-octyl. Cyclopentyl and cyclohexyl are preferredcycloalkyl groups.

The spacer length is the number of atoms of the spacer, counted alongthe shortest chain between the oligosaccharide residue and the peptidepart of the molecule, not counting the oxygen atom of theoligosaccharide residue which is connected to the spacer.

The term “prodrug” means a compound of the invention in which forinstance the amino group of the amidino-moiety is protected, e.g. byhydroxy or a (1-6C)alkoxycarbonyl group.

Solvates according to the invention include hydrates.

With regard to the synthetic way in which the biotin residue is attachedto compounds of the formula I the chemical literature offers severalpossibilities which can be utilized and by which different sets ofprotective groups well known to a person skilled in the art can beemployed. The biotin residue, comprising a reactive group of forinstance the activated ester, maleimide, iodoacetyl or primary aminetype, will preferably be reacted with an amine functional group, or athiol functional group, or a carboxylic acid functional group, or analdehyde functional group, the reaction taking place according to theconditions described in the literature (cf. Savage et al., Avidin-BiotinChemistry: A Handbook; Pierce Chemical Company, 1992).

The biotin residue can for instance be bonded directly to the(negatively charged) oligosaccharide residue or via an optionallyN-(1-4C)alkylated amino functional group of a oligosaccharide-spacerresidue or via an optionally N-(1-4C)alkylated amino acid residue to anoptionally N-(1-4C)alkylated amine functional group of theoligosaccharide residue of the compound of formula I.

In another aspect of this invention the biotin residue can for instancebe bonded directly to residue A or via an optionally N-(1-4C)alkylatedamino functional group of a linking residue or via an optionallyN-(1-4C)alkylated amino acid residue to an optionally N-(1-4C)alkylatedamine functional group of residue A of the compound of formula I.

Yet in another aspect of this invention the biotin residue can forinstance be introduced stepwise by first bonding directly to residue Aor via an optionally N-(1-4C)alkylated amino functional group of a partof the spacer of formula I or via an optionally N-(1-4C)alkylated aminoacid residue to an optionally N-(1-4C)alkylated amine functional groupof residue A of the compound of formula I and second bonding directly toan oligosaccharide or via an optionally N-(1-4C)alkylated aminofunctional group of part of the spacer of formula I or via an optionallyN-(1-4C)alkylated amino acid residue to an optionally N-(1-4C)alkylatedamine functional group of the (negatively charged) oligosaccharide ofthe compound of formula I, or vice versa.

In another aspect of the invention optionally N-alkylated amino acidresidues or α-N-substituted (beta-)amino acid analogues such asdescribed in [Bioconjugate Chem., Vol. 12, No. 4, 2001, 616-623] may beintroduced by a peptide coupling using methods known in the art. Theazido group is a suitable latent amine functional group which can beused in precursors of the compound of the formula I for the subsequentintroduction of the biotin residue.

A preferred process for the preparation of the compound of formula Icomprises a step wherein the benzamidine moiety in the residue A is inthe form of a precursor, preferably being the 1,2,4-oxadiazolin-5-onegroup, and is subsequently converted into the benzamidine bydeprotection, in particular by hydrogenation (Bolton, R. E. et al,Tetrahedron Letters, Vol 36, No 25, 1995, pp 4471-4474).

The compounds of the present invention are further prepared byderivatizing NAPAP (or a NAPAP-analogue) at the glycine position withcysteine or lysine or aspartate using methods generally known in theart, which compound subsequently (a) is coupled to aoligosaccharide-spacer residue or (b) is coupled to a spacer, which thenis derivatized with a thiol functional group or a carboxylic acidfunctional group and subsequently is coupled to an oligosaccharideresidue. Any suitable oligosaccharide may be used for this purpose, forexample oligosaccharides known in literature (e.g. from EP 0,454,220 andEP 0,529,715, but not limited to these sources) or commerciallyavailable oligosaccharides. The coupling of the spacer to theoligosaccharide can for instance be performed by using the methodsdescribed in EP 0,649,854.

The peptide coupling, a procedural step in the above described method toprepare the compounds of the invention, can be carried out by methodscommonly known in the art for the coupling—or condensation—of peptidefragments such as by the azide method, mixed anhydride method, activatedester method, the carbodiimide method, or, preferably, under theinfluence of ammonium/uronium salts like TBTU, especially with theaddition of catalytic and racemisation suppressing compounds likeN-hydroxysuccinimide, N-hydroxybenzotriazole and7-aza-N-hydroxybenzotriazole. Overviews are given in The Peptides,Analysis, Synthesis, Biology, Vol 3, E. Gross and J. Meienhofer, eds.(Academic Press, New York, 1981) and Peptides: Chemistry and Biology, N.Sewald and H.-D. Jakubke (Wiley-VCH, Weinheim, 2002).

Amine functions present in the compounds may be protected during thesynthetic procedure by an N-protecting group, which means a groupcommonly used in peptide chemistry for the protection of an α-aminogroup, like the tert-butyloxycarbonyl (Boc) group, the benzyloxycarbonyl(Z) group, the 9-fluorenylmethyloxycarbonyl (Fmoc) group or thephthaloyl (Phth) group, or may be introduced by demasking of an azidemoiety. Overviews of amino protecting groups and methods for theirremoval is given in the above mentioned The Peptides, Analysis.Synthesis, Biology, Vol 3 and Peptides: Chemistry and Biology.

The compounds of the invention, which can occur in the form of a freebase, may be isolated from the reaction mixture in the form of apharmaceutically acceptable salt. The pharmaceutically acceptable saltsmay also be obtained by treating the free base of formula (I) with anorganic or inorganic acid such as hydrogen chloride, hydrogen bromide,hydrogen iodide, sulfuric acid, phosphoric acid, acetic acid, propionicacid, glycolic acid, maleic acid, malonic acid, methanesulphonic acid,fumaric acid, succinic acid, tartaric acid, citric acid, benzoic acid,ascorbic acid and the like.

The compounds of this invention or intermediates thereof possess chiralcarbon atoms, and may therefore be obtained as a pure enantiomer, or asa mixture of enantiomers, or as a mixture containing diastereomers.Methods for obtaining the pure enantiomers are well known in the art,e.g. crystallization of salts which are obtained from optically activeacids and the racemic mixture, or chromatography using chiral columns.For diastereomers straight phase or reversed phase columns may be used.

The compounds of the invention may be administered enterally orparenterally. The exact dose and regimen of these compounds andcompositions thereof will necessarily be dependent upon the needs of theindividual subject to whom the medicament is being administered, thedegree of affliction or need and the judgment of the medicalpractitioner. In general, parenteral administration requires lowerdosages than other methods of administration which are more dependentupon absorption. However, the daily dosages are for humans preferably0.001-100 mg per kg body weight, more preferably 0.01-10 mg per kg bodyweight.

The medicament manufactured with the compounds of this invention mayalso be used as adjuvant in acute anticoagulant therapy. In such a case,the medicament is administered with other compounds useful in treatingsuch disease states.

Mixed with pharmaceutically suitable auxiliaries, e.g. as described inthe standard reference, Gennaro et al., Remington's PharmaceuticalSciences, (18th ed., Mack Publishing Company, 1990, see especially Part8: Pharmaceutical Preparations and Their Manufacture) the compounds maybe compressed into solid dosage units, such as pills, tablets, or beprocessed into capsules or suppositories. By means of pharmaceuticallysuitable liquids the compounds can also be applied in the form of asolution, suspension, emulsion, e.g. for use as an injectionpreparation, or as a spray, e.g. for use as a nasal spray.

For making dosage units, e.g. tablets, the use of conventional additivessuch as fillers, colorants, polymeric binders and the like iscontemplated. In general any pharmaceutically acceptable additive whichdoes not interfere with the function of the active compounds can beused.

Suitable carriers with which the compositions can be administeredinclude lactose, starch, cellulose derivatives and the like, or mixturesthereof, used in suitable amounts.

LEGENDS TO FIGURES

FIG. 1. Mean plasma levels determined by measurement of the anti-Xa oranti-IIa activity after s.c. administration of 100 nmol/kg of thebiotinylated compound III of this invention (“biotin”). Besides theplasma levels are given of Org 42675 (“original”) after determination ofthe anti-Xa activity.

FIG. 2. Shows the mean plasma levels±s.e.m. after s.c. administration of100 nmol/kg compound. At t=2 h avidin (10 mg/kg) was administered i.v.to those rats treated with compound III of this invention (“biotin”) orOrg 42675 (“original”). The pharmacokinetic behavior of compound III ofthis invention is effected by administration of Avidin in contrast tothe behavior of the original compound.

The invention is further illustrated by the following examples.

EXAMPLES Abbreviations Used

Aq.=aqueousATIII=antithrombin IBoc=tert-butyloxycarbonylDCM=dichloromethane

DiPEA=N,N-diisopropylethylamine DMF=N,N-dimethylformamide

Et=ethylfmoc=9-fluorenylmethoxycarbonylNMM=N-methyl morpholineMe=methylsat.=saturatedPRP=platelet rich plasmaPPP=platelet poor plasmaRT=room temperatureTBTU=2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborateTFA=trifluoroacetic acidTHF=tetrahydrofuran

Example 1 Scheme 1N^(ε)-(D-(+)-biotinyl)-N-{4-[[4-[[(1R)-1-[[4-(1,2,4-oxadiazol-5-onyl)phenyl]methyl]-2-oxo-2-(1-piperidinyl)ethyl]amino]-3-[[(4-methoxy-2,3,6-trimethylphenyl)sulfonyl]amino]-1,4-(S)-dioxo-butyl]amino]-butanoyl}-L-lysine(3)

Compound 2 (0.20 g, 0.27 mmol), which was prepared as described in WO01/42262, was dissolved in DCM (10 mL). DiPEA (0.14 mL, 0.81 mmol) wasadded followed by TBTU (86 mg, 0.27 mmol). After 1 h stirring under anatmosphere of nitrogen, compound 1 (N^(ε)-fmoc-Lys-OH, 0.27 mmol, Fluka)was added as a solid. DMF (5 mL) was added to dissolve the(N^(ε)-fmoc-Lys-OH. The mixture was stirred for 16 h and was poured in a0.5N HCl-solution. The solution was extracted with DCM (3×). Thecombined organic layers were washed with brine, dried (MgSO₄) andconcentrated under reduced pressure (850 mbar, 45° C.). The crudeproduct was purified by silica column chromatography (DCM/MeOH/AcOH,99/0/1→89/10/1, v/v/v), Remaining AcOH was removed by repeatedconcentration in toluene. Further purification was accomplished by HPLCchromatography (ACN/H₂O/0.1N TFA, 20:78:3→95:2:3) to give 0.15 g (53%)of the pure compound. TLC: Rf 0.5 (DCM/MeOH/AcOH, 90/9/1, v/v/v). LC-MS:m/z 1089 [M+H]¹⁺.

The fmoc protected intermediate (0.15 g, 0.14 mmol) was dissolved in THF(5 mL) and Et₂NH (2 mL) was added. The mixture was allowed to stir for45 minutes at 45° C. The solution was concentrated under reducedpressure and concentrated in toluene. ESI-MS: m/z 857 [M+H]¹⁺.

The N^(ε)-deprotected lysine derivative (0.14 mmol) was dissolved in DMF(3 mL) and was added to a solution of D-(+)-biotin (34 mg, 1 equiv.),TBTU (45 mg, 1 equiv.) and DiPEA (61 uL, 2.5 equiv.) in DMF (4 mL) thathad been stirred under a nitrogen atmosphere for 1 h. The resultingmixture was stirred for 16 h. The solvent was removed under reducedpressure. EtOAc (30 mL) was added and stirred. The solid product 3 wascollected by filtration and was washed with MeOH and EtOAc and dried invacuo. Yield 86 mg (57%). Rf 0.15 (DCM/MeOH, 9/1, v/v). ESI-MS: 1083.6[M+H]⁺.

General Procedure for Preparation of Compounds III and IV:

The carboxylic acid derivative (33 μmol) (i.e. compound 3 or 11) wasdried by repeated concentration in dry DMF (2×2 mL), dissolved in DMF (1mL) and stirred in the presence of TBTU (11 mg, 33 μmol) and DiPEA (5.7μL, 33 μmol), under an atmosphere of N₂. After 1 h, pentasaccharide 4(31 μmol) was added. The reaction mixture was stirred for 16 h at RT andanalyzed by ion exchange (Mono-Q) chromatography. The reaction mixturewas concentrated (<50° C., 15 mmHg). The (crude) product (10 mg/mL inH₂O/t-BuOH, 1/1, v/v) was deprotected by hydrogenation (H₂) over 10%Pd/C (an equal amount in weight was added with respect to the crudeproduct). After 16 h the solution was degassed, filtered over a 0.45 μMHPLC filter and concentrated under reduced pressure (<50° C., 15 mmHg).The conjugate was purified by ion exchange chromatography (Q-sepharose,buffer: H₂O→2M NaCl), followed by desalting with a Sephadex G25-column(H₂O) and lyophilization.

MethylO-2,3-di-O-methyl-4-O-<<<12-N-<<N^(ε)-(D-(+)-biotinyl)-N-<)]-{4-[[4-[[(1R)-1-[[4-(aminoiminomethyl)phenyl]methyl]-2-oxo-2-(1-piperidinyl)ethyl]amino]-3-[[(4-methoxy-2,3,6-trimethylphenyl)sulfonyl]amino]-1,4-(S)-dioxo-butyl]amino]-butanoyl}-L-lysyl>>-12-aza-3,6,9-trioxa-dodecyl>>>-6-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-(1->4)-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-alpha-L-idopyranuronosyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-alpha-D-glucopyranosideoctakis sodium salt (III)

Compound III was prepared and purified by conjugation of compound 3 (86mg, 81 μmol) with compound 4 (0.14 g, 80 μmol), which was prepared asdescribed in WO 01/42262, according to the general procedure. Yield 0.14g (60%). ESI/TOF-MS: 880.91 [M−3H]³⁻, 888.57 [M−3H+Na]³⁻, 660.43[M−4H]⁴⁻.

1H-NMR (D₂O, 600 MHz); reference water signal at 4.71 ppm hampersreliable integration of signals between 4.80-4.50 ppm. δ 7.66 (m, 2H),7.30 (m, 2H), 6.74 (m, 1H), 5.36 (m, 1H), 5.25 (m, 1H), 5.05 (m, 1H),4.98 (m, 1H), 4.84 (m, 1H), 4.46 (m, 2H), 4.31 (m, 1H), 4.27-4.13 (5H),4.13-3.99 (5H), 3.92 (m, 1H), 3.88-3.70 (8H), 3.70-3.07 (49H±5),3.07-2.92 (4H), 2.85 (m, 1H), 2.76 (m, 1H), 2.65 (m; 1H), 2.51 (s, 3H),2.40 (s, 3H), 2.36-2.22 (m, 2H), 2.21-2.11 (m, 2H), 2.08 (m, 1H),2.06-1.91 (4H), 1.83-1.03 (20H±2).

Example 2 Scheme 2 tert-Butyl15-N-(9-fluorenylmethyloxycarbonyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate(6)

tert-Butyl 15-amino-3,6,9,12-tetraoxa-pentadecanoate (5) (0.50 g, 1.45mmol) was dissolved in THF (7.5 mL) and H₂O (5 mL). 4 N NaOH solutionwas added until pH was approximately 9. N-9-Fluorenylmethylcarbamate-succinimide (FmocOSu, 0.54 g, 1.60 mmol, 1.1 eq) was added inportions. After 10 min. additional 4 N NaOH solution was added to adjustthe pH to approximately 9. After 3 h the reaction mixture was acidifiedwith 1 N HCl solution to pH 6-7. H₂O was added to the reaction mixturewhich was then extracted 3 times with EtOAc. The organic phase waswashed with brine and dried over MgSO₄. After filtration the solvent wasremoved under reduced pressure (50 mbar, 50° C.). The crude oil waspurified by silica column chromatography (DCM/MeOH, 1/0→95/5, v/v), togive compound 6 as a yellowish oil (0.61 g, 79%). Rf 0.64 (DCM/MeOH,95/5, v/v).

15-N-(9-Fluorenylmethyloxycarbonyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoate(7)

Compound 6 was dissolved in DCM (3.5 mL) and TFA (3.5 mL) was addedunder a nitrogen atmosphere. After 1.5 h of stirring the reactionmixture was concentrated under reduced pressure. Then the excess of TFAwas removed by repeated concentration in toluene. DCM/Et₂O (100 mL, 1/2,v/v) was added and washed with 1 N HCl. The water layer was extractedwith DCM/Et₂O (100 mL, 1/2, v/v). The combined organic layers werewashed with brine and dried over MgSO₄. After filtration the solvent wasremoved under atmospheric pressure (50° C.). The crude oil was purifiedby silica column chromatography (DCM/MeOH/AcOH, 99/0/14-89/10/1, v/v/v),to give compound 7. Remaining AcOH was removed by dissolving the crudeoily product in DCM/Et₂O (1/2, v/v) and washing with H₂O (3 times) andbrine followed by drying over MgSO₄. After filtration the solvent wasremoved under atmospheric pressure (50° C.) to give compound 7 as ayellowish oil (0.37 g, 67%). Rf 0.32 (DCM/MeOH, 89/10/1, v/v).

tert-Butyl15-N-(9-Fluorenylmethyloxycarbonyl)-15-aza-3,6,9,12-tetraoxa-pentadecanoyl-ε-N-(Z)-L-lysine(8)

Compound 7 (0.37 mg, 0.77 mmol) was dissolved in DCM (18 mL). DIPEA(0.40 μL, 2.31 mmol, 3 eq) and TBTU (0.25 g, 0.77 mmol) weresubsequently added under an atmosphere of N₂ and the solution wasallowed to stir for 10 min. Then ε-(Z)-L-Lys-OtBu.HCl (0.29 g, 0.77mmol) was added and the mixture was stirred for an additional 1.5 h. Thereaction mixture was diluted with DCM and rinsed with H₂O, 0.1 N HCl,sat. NaHCO₃-sol. and brine. The organic phase was dried (MgSO₄) andconcentrated under atmospheric pressure. Purification was effected bysilica gel column chromatography (DCM/MeOH, 1/0-9/1, v/v), to givecompound 8 as a yellowish oil (0.51 g, 83%). Rf 0.85 (DCM/MeOH, 9/1,v/v). ESI-MS: 792.6[M+H]⁺, 814.6 [M+Na]⁺, 736.4 [M−tBu+H]⁺

tert-Butyl15-N-tert-butyloxycarbonyl-15-aza-3,6,9,12-tetraoxa-pentadecanoyl-ε-N-(Z)-L-lysine(9)

Compound 8 (0.26 g, 0.32 mmol) was dissolved in THF (5 mL). Et₂N (1 mL)was added and the solution was allowed to stir for 24 h. The excess Et₂Nand solvent were removed under reduced pressure (50° C.). Toluene wasadded and removed under reduced pressure (50° C., 65 mbar) to give theN-deprotected intermediate product (0.21 g, 0.32 mmol), Rf 0.23(DCM/MeOH, 9/1, v/v) ESI-MS: 570.4 [M+H]⁺, 514.4 [M−tBu+H]⁺. The crudeproduct was dissolved in DCM (3 mL). Et₃N (0.11 mL) was added followedby di-tert-butyl dicarbonate (73 mg, 0.34 mmol, 1.1 eq) under anatmosphere of N₂. After stirring for 5 h the mixture was added to a cold(5° C.) solution of 0.1 N HCl and extracted with EtOAc. The organiclayer was washed with brine and dried (MgSO₄). After filtration thesolvents were removed under reduced pressure (180 mbar, 50° C.).Purification was effected by silica gel column chromatography (DCM/MeOH,1/0-95/5, v/v), to give 9 as a colorless oil (0.17 g, 82%). Rf 0.5(DCM/MeOH, 9/1, v/v). ESI-MS: 670.6[M+H]⁺, 692.4[M+Na]⁺, 570.4[M−Boc+H]⁺, 514.1 [M−Boc−tBu+H]⁺.

15-aza-3,6,9,12-tetraoxa-pentadecanoyl-ε-[D-(+)-biotinyl]-L-lysine (10)

Compound 9 (0.23 g, 0.34 mmol) was dissolved in EtOH (8 mL) and H₂O (1.2mL). After flushing the solution with nitrogen for 5 minutes, Pd/C 10%(0.11 g) was added. Hydrogen was passed trough the solution for 4 h.Nitrogen was flushed trough the solution for 10 minutes to remove allhydrogen. The mixture was filtered over decalite and was concentratedunder reduced pressure (170 mbar, 50° C.) to give the N-L-lysinedeprotected intermediate as a colorless oil (0.15 g, 81%). Rf 0.02(DCM/EtOAc, 9/1, v/v).

D-(+)-Biotine (75 mg, 0.31 mmol) was suspended in DCM (7 mL). DIPEA(0.11 mL, 0.62 mmol, 2 eq) and TBTU (0.10 g, 0.31 mmol) weresubsequently added under an atmosphere of N₂ and the solution wasallowed to stir for 1 h. A solution of the above described N-L-lysinedeprotected intermediate in DCM (3 mL) was added to the reactionmixture. The mixture was allowed to stir for 16 h. H₂O was added andextracted with DCM (3×). The organic layer was dried (MgSO₄), filteredand concentrated under reduced pressure (850 mbar, 50° C.). Purificationwas effected by silica gel column chromatography (eluens: DCM/MeOH,1/0→9/1 v/v), to give an oil (0.13 g, 60%). Rf 0.48 (DCM/MeOH, 9/1,v/v). ESI-MS: 762.6[M+H], 784.6[M+Na], 662.4 [M−Boc+H], 606.4[M−Boc−tBu+H]. The oil was dissolved in a dry 4 N HCl solution indioxane (4 mL) and stirred. After 1 h an insoluble oil appeared afterwhich the solvent was removed under reduced pressure (100 mbar, 50° C.)to give compound 10 in quantitative yield. ESI-MS: 606.4 [M+H]⁺, 628.4[M+Na]⁺.

Scheme 3N^(ε)-(D-(+)-biotinyl)-N-{{4-[[4-[[(1R)-1-[[4-(1,2,4-oxadiazol-5-onyl)phenyl]methyl]-2-oxo-2-(1-piperidinyl)ethyl]amino]-3-[[(4-methoxy-2,3,6-trimethylphenyl)sulfonyl]amino]-1,4-(S)-dioxo-butyl]amino]-butanoyl}-15-N-(15-aza-1-oxo-3,6,9,12-tetraoxa-pentadecyl)]}-L-lysine(11)

Compound 10 (0.12 g, 0.21 mmol) was coupled to compound 2 (0.15 g, 0.21mmol) as described for the preparation of compound 3. The crude productwas purified by preparative HPLC (C18, ACN/H₂O, 0.01% TFA) to givecompound 11 in pure form. Yield 60 mg (22%). ESI-MS: 1316.8 [M+H]⁺

Methyl.O-2,3-di-O-methyl-4-O-<<<12-N-<<N^(ε)-(D-(+)-biotinyl)-N-<[15-N-(15-aza-1-oxo-3,6,9,12-tetraoxa-pentadecyl)]-{4-[[4-[[(R)-1-[[4-(aminoiminomethyl)phenyl]methyl]-2-oxo-2-(1-piperidinyl)ethyl]amino]-3-[[(4-methoxy-2,3,6-trimethylphenyl)sulfonyl]amino]-1,4-(S)-dioxo-butyl]amino]-butanoyl}>-L-lysyl>>-12-aza-3,6,9-trioxa-dodecyl>>>-6-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-beta-D-glucopyranuronosyl-(1->4)-O-2,3,6-tri-O-sulfo-alpha-D-glucopyranosyl-(1->4)-O-2,3-di-O-methyl-alpha-L-idopyranuronosyl-(1->4)-3-O-methyl-2,6-di-O-sulfo-alpha-D-glucopyranosideoctakis sodium salt (IV)

Compound 11 was coupled to compound 4 and the intermediate conjugate wasdeprotected according to the general procedure to give compound IV.

Yield 9.2 mg (7.6%)

1H-NMR (D2O, 600 MHz), reference water signal at 4.70 ppm hampersreliable integration of signals between 4.80-4.54 ppm. δ 7.59 (d, 2H),7.23 (d, 2H), 6.68 (s, 1H), 5.32 (m, 1H), 5.19 (m, 1H), 4.91 (m, 1H),4.85 (m, 1H), 4.76 (m), 4.53-4.31 (3H), 4.30-4.05 (9H), 4.04-3.90 (7H),3.89-3.62 (7H), 3.61-3.42 (42H±4), 3.42-3.31 (18H±2), 3.31-3.14 (12H),3.14-3.05 (5H), 3.03-2.94 (3H), 2.93-2.84 (2H), 2.81 (dd, 1H), 2.70 (dd,1H), 2.59 (d, 1H), 2.44 (s, 3H), 2.34 (s, 3H), 2.22-2.08 (4H), 2.06 (t,2H), 1.96 (s, 3H), 1.72-1.01 (18H±2)

ESI/TOF-MS: m/z 574.72 [M−5H]⁵⁻, at m/z 718.66 [M−4H]⁴⁻, at m/z 958.56[M−3H]³⁻

Example 3 Pharmacology In Vitro Test for Determination of theAnti-Factor Xa and Factor IIa Activity in Human Plasma

The anti-factor Xa and IIa activity of the tested compounds in humanplasma were measured amidolytically using S2222 or S2238 (Chromogenix,Chromogenics Ltd, Molndal, Sweden) as substrates, respectively. Theprotocols were based on the method described by Teien and Lie. (Teien AN, Lie M. Evaluation of an amidolytic heparin assay method increasedsensitivity by adding purified antithrombin III. Thromb. Res. 1977, 10:399-410). Both activities are expressed in IC-50 (Mol/L).

TABLE 1 Summary of in vitro antithrombotic activities Compound III ofCompound ORG 42675 this invention Anti-IIa 1.78E−08 1.62E−08 Humanplasma pH 7.4 IC-50 (M) Anti-Xa 7.67E−10 8.43E−10 Human plasma pH 7.4IC-50 (M)

3.1 Pharmacokinetics

The pharmacokinetic properties of Org 42675 and of compound III of thisinvention were studied in male Wistar rats of 300-400 gr. The rats wereanaesthetized by inhalation of a mixture of O₂/N₂O/isoflurane, afterwhich the right jugular vein was cannulated. The next day rats weretreated s.c. with doses of 100 nmol/kg. After s.c. administration, bloodwas sampled at several time intervals. Then the blood was centrifugedafter which the plasma was siphoned off and stored at −20° C. until use.The concentration of the tested compound was measured amidolyticallyusing S-2222 or S-2238 as substrates (Chromogenix, Chromogenics Ltd,Molndal, Sweden) to determine the anti-Xa or anti-IIa activity,respectively. Both procedures were based on the methods of Teien and Lie(Teien A N, Lie M. Evaluation of an amidolytic heparin assay methodincreased sensitivity by adding purified antithrombin II. Thromb. Res.1977, 10: 399-410). The concentrations in the obtained plasma sampleswere determined against a calibration curve which was made of the stocksolution of the tested compound itself. The concentration in the sampleswas expressed in nmol/mL and the kinetic parameters were calculated withthe noncompartment model of WinNonlin. (see FIG. 1)

TABLE 2 Pharmacokinetic parameters after s.c. administration of compoundIII of this invention or Org 42675 (100 nmol/kg) in rat. Compound III ofCompound III of this invention this invention Org 42675 Mean ± s.e.m.Mean ± s.e.m. Mean ± s.e.m. anti_Xa anti-IIa anti_Xa Tmax (h)  1.2 ±0.4. 1.5 ± 0.5 2.3 ± 0.2 Cmax (nmol/mL) 1.1 ± 0.1 1.1 ± 0.3  1.0 ± 0.02T½ eli (h) 3.9 ± 0.3 2.7 ± 0.5 2.7 ± 0.2 AUCinf 7.2 ± 0.2 5.0 ± 0.6 5.3± 0.3 (h · nmol/mL) Vz (mL/kg)  78 ± 4.6  78 ± 4.2  74 ± 4.5 Cl(mL/h/kg) 13.9 ± 0.4  20.6 ± 2.4  19.1 ± 1.1  MRT (h) 5.9 ± 0.5 4.3 ±0.3 4.6 ± 0.2 Experiment performed in n = 3/treatment.It is concluded that within the variability of the experiment compoundIII of this invention and Org 42675 show the same pharmacokineticbehavior in rats.

3.2 Pharmacokinetics—Neutralization Experiment:

Rats were treated with compound III of this invention, or Org 42675 at adose of 100 nmol/kg s.c. At t=2 h, a blood sample was taken and 10 mg/kgof Avidin (from egg white, Sigma) was administered i.v. to the ratstreated with compound III of this invention or Org 42675. Blood wassampled at 0.17, 0.5, 1, 2, 3, and 7 hours subsequently. The blood wastreated as described in the pharmacokinetic experiment and theconcentration of the samples was determined by measuring the (residual)anti-Xa or anti-IIa activity. (see FIG. 2)

TABLE 3 The area under the curves (AUC's) calculated after s.c.administration of 100 nmol/kg of compound III of this invention or Org42675 and avidin (10 mg/kg) at t = 2 h. Compound III of Compound III ofCompound this invention this invention Org 42675 Mean ± s.e.m. Mean ±s.e.m. Mean ± s.e.m. anti_Xa anti-IIa anti_Xa AUCinf 1.2 ± 0.1 0.8 ± 0.14.8 ± 0.6 (h · nmol/mL) Data calculated from T = 2 h. Experimentperformed in n = 3/treatment.

It is concluded that after s.c. administration of compound III of thisinvention (100 nmol/kg), the antithrombotic activity as determined bymeasuring the (residual) anti-Xa and anti-IIa activity can beneutralized by administration of 10 mg/kg i.v. of avidin. Theneutralization of compound III of this invention by avidin is reflectedby the is reflected by the rapid reduction of its plasma concentrationand the strongly reduced AUCinf. of compound III of this invention afteradministration of Avidin in comparison to compound Org 42675.Furthermore, the pharmacokinetic behavior of the non-biotinylatedequivalent compound Org 42675 is not affected by the addition of avidin.The latter confirms that the neutralization is associated with thepresence of the biotin label and that it does not affect thepharmacokinetic behavior of the dual inhibitor.

1-25. (canceled)
 26. An antithrombotic compound of the formula (I)oligosaccharide-spacer-A  (I), wherein the oligosaccharide is anegatively charged pentasaccharide of the formula (II)

wherein R⁵ is OSO₃ ⁻ or (1-8C)alkoxy, the charge being compensated bypositively charged counterions; the spacer is an essentiallypharmacologically inactive flexible linking residue having a chainlength of 10 to 70 atoms; A is the residue—CH[NH—SO₂—R¹][CO—NR²—CH(4-benzamidine)-CO—NR³R⁴], wherein R¹ is phenyl,naphthyl, 1,2,3,4-tetrahydronaphthyl, (iso)quinolinyl,tetrahydro(iso)quinolinyl, 3,4-dihydro-1H-isoquinolinyl, chromanyl orthe camphor group, which groups may optionally be substituted with oneor more substituents selected from (1-8C)alkyl or (1-8C)alkoxy; andwherein R² and R³ are independently H or (1-8C)alkyl; R⁴ is (1-8C)alkylor (3-8C)cycloalkyl; or R³ and R⁴ together with the nitrogen atom towhich they are bonded are a nonaromatic (4-8)membered ring optionallycontaining another heteroatom, the ring optionally being substitutedwith (1-8C)alkyl or SO₂-(1-8C)alkyl; or a pharmaceutically acceptablesalt thereof; wherein the spacer of the compound of formula I comprisesat least one covalent bond with a biotin residue or an analogue of theformula —(CH₂)₄—NR-BT, where BT is the residue

where R is H or (1-4C)alkyl.
 27. The compound of claim 26, wherein thepentasaccharide residue has the structure

wherein R⁵ is OCH₃ or OSO₃ ⁻.
 28. The compound of claim 27, wherein thepentasaccharide residue has the structure


29. The compound of claim 26, wherein R¹ is phenyl or naphthyl,optionally substituted with one or more substituents selected frommethyl or methoxy.
 30. The compound of claim 29, wherein R¹ is4-methoxy-2,3,6-trimethylphenyl.
 31. The compound of claim 26, whereinNR³R⁴ represents the piperidinyl group.
 32. The compound of claim 26,wherein R² is H.
 33. The compound of claim 26, where the compound is

or a pharmaceutically acceptable salt thereof.
 34. The compound of claim33, where the compound is in the form of a sodium salt.
 35. The compoundof claim 26 wherein the spacer has a chain length of 10-50 atoms. 36.The compound of claim 35 wherein the spacer has a chain length of 16-22atoms.
 37. The compound of claim 36 wherein the spacer has a chainlength of 19 atoms.
 38. The compound of claim 36 wherein the spacercomprises at least one —(CH₂CH₂O)-element.
 39. The compound of claim 37wherein the spacer comprises at least one —(CH₂CH₂O)-element.
 40. Amethod for treating thrombosis in a subject in need of such treatment,the method comprising administering to the subject an effective amountof an antithrombotic compound of the formula (I)oligosaccharide-spacer-A  (I), wherein the oligosaccharide is anegatively charged pentasaccharide of the formula (II

wherein R⁵ is OSO₃ ⁻ or (1-8C)alkoxy, the charge being compensated bypositively charged counterions; the spacer is an essentiallypharmacologically inactive flexible linking residue having a chainlength of 10 to 70 atoms; A is the residue—CH[NH—SO₂—R¹][CO—NR²—CH(4-benzamidine)-CO—NR³R⁴], wherein R¹ is phenyl,naphthyl, 1,2,3,4-tetrahydronaphthyl, (iso)quinolinyl,tetrahydro(iso)quinolinyl, 3,4-dihydro-1H-isoquinolinyl, chromanyl orthe camphor group, which groups may optionally be substituted with oneor more substituents selected from (1-8C)alkyl or (1-8C)alkoxy; andwherein R² and R³ are independently H or (1-8C)alkyl; R⁴ is (1-8C)alkylor (3-8C)cycloalkyl; or R³ and R⁴ together with the nitrogen atom towhich they are bonded are a nonaromatic (4-8)membered ring optionallycontaining another heteroatom, the ring optionally being substitutedwith (1-8C)alkyl or SO₂-(1-8C)alkyl; or a pharmaceutically acceptablesalt thereof; wherein the spacer of the compound of formula I comprisesat least one covalent bond with a biotin residue or an analogue of theformula —(CH₂)₄—NR-BT, where BT is the residue where R is H or(1-4C)alkyl,

and neutralizing the anti-thrombotic activity of the compound byadministering avidin or streptavidin.
 41. The method of claim 40,wherein the pentasaccharide residue has the structure

wherein R⁵ is OCH₃ or OSO₃ ⁻.
 42. The method of claim 41, wherein thepentasaccharide residue has the structure


43. The method of claim 40, wherein R¹ is phenyl or naphthyl, optionallysubstituted with one or more substituents selected from methyl ormethoxy.
 44. The method of claim 43, wherein R¹ is4-methoxy-2,3,6-trimethylphenyl.
 45. The method of claim 40, whereinNR³R⁴ represents the piperidinyl group.
 46. The method of claim 40,wherein R² is H.
 47. The method of claim 40, where the compound is

or a pharmaceutically acceptable salt thereof.
 48. The method of claim47, where the compound is in the form of a sodium salt.
 49. The methodof claim 40 wherein the spacer has a chain length of 10-50 atoms. 50.The method of claim 49 wherein the spacer has a chain length of 16-22atoms.
 51. The method of claim 50 wherein the spacer has a chain lengthof 19 atoms.
 52. The method of claim 50 wherein the spacer comprises atleast one —(CH₂CH₂O)-element.
 53. The method of claim 51 wherein thespacer comprises at least one —(CH₂CH₂O)-element.